Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.
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PMID:Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. 12 60

Splicing factor SF1 represents one of the proteins that function early in the splicing of nuclear pre-mRNA in the formation of a presplicing complex. SF1 was purified to homogeneity from HeLa cell nuclear extracts by column chromatography. It consists of a single polypeptide of 75 kDa and is distinct from other protein factors that function early in spliceosome assembly. SF1 activity is completely resistant to temperatures of up to 100 degrees C. The purified protein does not appear to be associated with RNA-binding, RNA-annealing, or ATPase activity.
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PMID:Purification of splicing factor SF1, a heat-stable protein that functions in the assembly of a presplicing complex. 140 44

1. o-Iodosobenzoic acid (IOB) caused the formation of a disulfide bridge between SH1 and SH2 groups of myosin SF1 rendering inactive its ATPase activity. 2. IOB at high concentrations provoked fragmentation of SF1 at its tryptophan residues. 3. The main fragmentation point was located at 15 K from the amino terminus of the myosin heavy chain. 4. Actin was not fragmented by IOB. It protected SF1 tryptophans from IOB attack. 5. These results suggest a possible use of IOB as a reagent to study protein tryptophan under nondenaturing conditions.
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PMID:o-Iodosobenzoic oxidation and cleavage of myosin subfragment 1. 158 26

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (A1) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under nondenaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 degrees C or 37 degrees C with free A1 or A2 to form the subfragment 1 isozymes SF1(A1) or SF1(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding domain.
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PMID:The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity. 611 11

The obliquely striated body wall muscle of the earthworm Lumbricus terrestris L. possesses a dual actin-linked and myosin-linked regulatory system. Tropomyosin from this muscle has now been purified and its functional properties compared to tropomyosin from vertebrate skeletal muscle. Earthworm tropomyosin has a molecular weight of about 70 000 and is composed of two polypeptide chains of molecular weight of 34 000 and 37 000. Structural and functional similarities to skeletal muscle tropomyosin were demonstrated with respect to the formation and periodicity of paracrystals and nets and the potentiation of skeletal muscle acto-SF1 ATPase activity at low ATP concentration. Likewise, earthworm tropomyosin inhibited skeletal muscle acto-HMM ATPase activity at normal ATP concentrations but to a much greater extent than skeletal muscle tropomyosin; this inhibition was removed by skeletal muscle troponin, in the presence of Ca2+. In a system containing earthworm myosin and skeletal muscle actin, earthworm tropomyosin had no detectable influence on the actin-activated ATPase activity. It is concluded that earthworm tropomyosin plays an active role in the actin-linked troponin-dependent regulatory system and has no measurable effect on the regulation via myosin.
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PMID:Properties of tropomyosin from the dual-regulated obliquely striated body wall muscle of the earthworm (Lumbricus terrestris L.). 621 Jul 9

The peripheral membrane portion (SF1) of proton-translocating ATPase of Salmonella typhimurium and its alpha, beta, and gamma subunits were purified and compared with the same portion (EF1) from Escherichia coli. The alpha, beta, and gamma subunits of these F1's were found to be mutually interchangeable, and all possible combinations of the three subunits from EF1 and SF1 showed ATPase activity. Both F1's could bind functionally to the integral membrane part (F0) of either bacterium, suggesting that F0 and F1 are interchangeable in these two bacteria and thus that the two F1's are closely similar at the level of subunit structure. However, SF1 differed from EF1 in some enzymological properties such as its specific activity and susceptibilities to sodium dodecyl sulfate and methanol. The specific ATPase activity of EF1 was more than twice that of SF1, and hybrid enzymes containing the beta subunit of EF1 had higher activity than other hybrids. Amino acid analysis suggested that the primary structures of the alpha subunits of the two F1's are less homologous than those of the beta subunits. Thus, the primary structure of the alpha subunit may be more species specific than that of the beta subunit.
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PMID:Comparison of F1's of oxidative phosphorylation from Escherichia coli and Salmonella typhimurium and demonstration of interchangeability of their subunits. 623 53

Evidence is presented that, under conditions of 4.7 M NH4Cl and 10 mM Mg-ATP where no subunit dissociation can be detected by transport methods, a dynamic equilibrium exists in subfragment 1 between the associated and dissociated subunits. This is readily discerned by the formation of hybrid subfragment 1 species when a subfragment 1 isozyme is incubated with excess free light chains of the alternate isozyme. A similar process occurs with p-N,N'-phenylenedimaleimide (pPDM)-modified subfragment 1 containing [14C]Mg-ADP, but in this case, although extensive amounts of hybrid are formed, no loss of the trapped nucleotide is observed. Subunit scrambling without loss of the trapped nucleotide is apparent from incubating pPDM-SF1(A2)-[14C]Mg-ADP with unmodified SF1(A1) under similar conditions since the mixture subsequently contains SF1(A1), SF1(A2)h, pPDM-SF1(A1)h-[14C]Mg-ADP and pPDM-SF1(A2)-[14C]Mg-ADP. These data show that the nucleotide trapped in the presumptive active site does not escape during the dissociation-reassociation cycle, and suggest that the ATPase site resides solely on the heavy chain.
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PMID:Subunit interactions in myosin subfragment 1. Subunit scrambling is independent of catalytic center involvement. 645 31

The formation of hybrid myosin and subfragment 1 species by incubation of these proteins with free alkali light chains at physiological ionic and temperature conditions is described. Exchange of bound alkali light chain on myosin by free alkali light chains under these conditions is readily demonstrated from the subunit composition of the isolated myosin. Therefore, the light chain exchange previously described for the one-headed subfragment 1 [Sivaramakrishnan, M., & Burke, M (1981) J. Biol. Chem. 256, 2607--2610] also occurs in the two-headed myosin molecule. It is found than the isozyme to hybrid transformation is dependent on both the temperature and the ionic strength of the incubation mixture but is relatively independent of pH in the range 6.5--8.0. A comparison of the SF1(A1) leads to SF1(A2)h system with the SF1(A2) leads to SF1(A1)h system indicates that more hybrid is formed in the latter case. With the assumption that hybrid formation reflects the degree of reversible dissociation exhibited by the isozyme, under the particular experimental condition employed, the data signify that the subunit interactions in the two isozymes are not identical and that the heavy chain--A1 interactions are significantly more stable that the heavy chain--A2 ones. An examination of the ATPase properties of the thermal hybrids in the presence and absence of actin indicates close similarities to their corresponding "native" isozymic counterparts.
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PMID:Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids. 645 38

The human coronavirus 229E replicase gene encodes a protein, p66HEL, that contains a putative zinc finger structure linked to a putative superfamily (SF) 1 helicase. A histidine-tagged form of this protein, HEL, was expressed using baculovirus vectors in insect cells. The purified recombinant protein had in vitro ATPase activity that was strongly stimulated by poly(U), poly(dT), poly(C), and poly(dA), but not by poly(G). The recombinant protein also had both RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. The DNA helicase activity of the enzyme preferentially unwound 5'-oligopyrimidine-tailed, partial-duplex substrates and required a tail length of at least 10 nucleotides for effective unwinding. The combined data suggest that the coronaviral SF1 helicase functionally differs from the previously characterized RNA virus SF2 helicases.
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PMID:The human coronavirus 229E superfamily 1 helicase has RNA and DNA duplex-unwinding activities with 5'-to-3' polarity. 1091

The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 bp duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths </=8 nt, even at high [UvrD]/[DNA] ratios. Unwinding is observed for DNA substrates with 3' ssDNA tail lengths >/=12 nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrD(tot)]/[DNA(tot)]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP. Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail.
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PMID:A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro. 1252 99


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